PROJECT SUMMARY Eukaryotic precursor mRNAs (pre-mRNAs) must undergo extensive processing, including splicing of intervening sequences (introns) and formation of the mRNA 3' end by cleavage and polyadenylation (CP), to produce a functional mRNA. The vast majority of human genes are alternatively spliced, generating multiple mRNAs from a single gene, and contain more than one CP site, enabling the production of mRNAs with different 3' ends. Changes in RNA processing are the basis of, or contribute to, a number of human diseases. Understanding the molecular mechanisms associated with RNA processing pathologies can provide key insights into normal processing mechanisms and may suggest new therapeutic approaches. Although originally thought to be distinct processes, a variety of studies have shown that alternative splicing (AS), mRNA 3' end formation and transcription are coupled to one another. However, the molecular basis for such coupling in general remains to be determined. Our long-term goal is to understand the detailed molecular mechanisms of pre-mRNA processing and elucidate how different pre-mRNA processing events are coordinated on a genome-wide level. The common overarching objective of the three specific aims of this application is to determine the mechanism(s) by which a specific pre-mRNA processing factor can regulate both AS and mRNA 3' end formation. Our work over the past funding period has revealed two novel examples of coupling between pre-mRNA splicing and mRNA 3' end formation that are subjects of this application. First, we have found, unexpectedly, that an oncogenic mutant of the essential splicing factor U2AF35, U2AF35(S34F), promotes transformation by altering the interaction between U2AF and cleavage factor Im (CFIm), a core component of the mRNA 3' end processing machinery, leading to increased use of distal CP sites. Second, we discovered a completely unanticipated role for the mRNA 3' end formation factors CPSF and SYMPK in global promotion of AS. Recently, we have found that YTHDC2, a reader protein of the mRNA modification N6 adenosine methylation (m6A), can promote AS and may also be involved in mRNA 3' end formation. In this application, we propose innovative experimental systems and approaches to determine: (1) how master regulators of splicing (U2AF) and CP site selection (CFIm) function coordinately, (2) the mechanistic basis by which the mRNA 3' end formation factor CPSF/SYMPK controls AS, and (3) the role of the m6A reader YTHDC2 in AS and mRNA 3' end formation. We believe the results of the proposed experiments will substantially advance the RNA processing field by providing an integrated view of processing activities that, until recently, were considered to be independent. By elucidating the detailed mechanisms by which different pre-mRNA processing events are coordinated, the experiments proposed in this application will establish new paradigms that have broad significance to the RNA processing field and for the dysregulation of gene expression that occurs in human diseases that are caused by alterations in RNA processing.